CN111000994A

CN111000994A - Liquid vaccine composition, preparation method and application thereof

Info

Publication number: CN111000994A
Application number: CN201911367536.6A
Authority: CN
Inventors: 王治伟; 范鑫; 徐树利; 隋晓斌; 吴慕胜; 李海涛; 高强; 尹卫东
Original assignee: Sinovac Research & Development Co ltd
Current assignee: Sinovac Research & Development Co ltd
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-04-14

Abstract

The present invention relates to a liquid vaccine composition comprising: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus Sabin strain I, inactivated poliovirus Sabin strain II, inactivated poliovirus Sabin strain III, a haemophilus influenzae type b capsular polysaccharide-protein conjugate. The liquid vaccine composition provided by the invention avoids the safety risk and the limitation of capacity caused by the freeze-drying process, avoids the complicated re-dissolving process of the freeze-dried vaccine before immunization, and reduces the production and use cost. Furthermore, the vaccine composition provided by the invention can be used for basic immunization or boosting immunization, and can improve the titer of the antibody in the serum of an immunized person on the basis of ensuring the safety.

Description

Liquid vaccine composition, preparation method and application thereof

Technical Field

The invention relates to the field of vaccines, and particularly relates to a liquid vaccine composition capable of preventing pertussis, diphtheria, tetanus, poliomyelitis and haemophilus influenzae type b at the same time.

Background

Timely vaccination is an effective preventative measure. Pertussis, diphtheria and tetanus combination vaccines (DTPs), the earliest vaccines incorporated into the WHO expanded immunization program (EPI), have played an important role in the prevention and control of these three infectious diseases. In the 70 s of the 20 th century, due to serious adverse reactions after whole cell pertussis vaccine (DTwP) inoculation, the phenomenon of resisting vaccination and non-use of vaccine appears in Japan, the Netherlands and other countries, the inoculation rate is reduced, and the disease rate of pertussis is rebounded. At present, most developed countries are prepared into cell-free diphtheria-pertussis-tetanus vaccine (DTaP) with small side reaction by co-purification or chromatographic purification, and the cell-free diphtheria-pertussis-tetanus vaccine is used as a basic combined vaccine and a conventional vaccine for immunization. Acellular pertussis vaccines can be divided into co-purified vaccines and chromatographically purified vaccines from the production process point of view. At present, some enterprises in China and Japan adopt a co-purification process, namely after bacterial culture, protective antigens such as PT, FHA, PRN and the like are precipitated by salting out, then impurities are removed by sucrose density gradient centrifugation, and meanwhile, effective components rich in PT, FHA and PRN are collected. The chromatographic purification is to purify different protective antigens respectively by adopting column chromatography, and then quantitatively proportioning each antigen to prepare the vaccine. The separation and purification method has higher cost, but has the advantages of definite components, easy quality control and less side effect. At present, most developed countries adopt column chromatography to separate and purify each component to produce acellular pertussis vaccines.

Poliomyelitis (WPV) is a widespread and extremely harmful acute infectious disease caused by polioviruses type I, II and III, which can affect people of any age group, but mainly children under 3 years old (accounting for 50% of all cases) and seriously cause irreversible paralysis of limbs and even death. Before the vaccine came into existence, the disease was prevalent worldwide. In the middle and late 50 s, Salk and Sabin, two scientists in the United states, succeeded in developing Inactivated Polio Vaccine (IPV) and Oral attenuated live polio Vaccine (OPV). Since then, people have had powerful weapons to prevent and destroy polio, and practice has proven that both vaccines are effective. The World Health Organization (WHO) initiated a worldwide initiative to eliminate poliomyelitis in 1988, after which activities to eliminate poliomyelitis have made major progress both in reducing the incidence of poliomyelitis and in reducing the spread of poliovirus. In 2007 from 1 month to 2008 from 6 months, 16 countries detected wild strains (WPV) from fecal specimens of AFP cases, only WPVI type and WPVIII type, and no WPVII type was detected worldwide since 1999, and these advances were made thanks to a polio eradication strategy that was commonly practiced throughout the world. In the action of eliminating poliomyelitis (WPV) globally, oral poliomyelitis attenuated live vaccine (OPV) is widely applied, and the incidence rate of the poliomyelitis globally is greatly reduced. However, due to the risk of Vaccine Associated Paralytic Poliomyelitis (VAPP), and Vaccine-derived Poliovirus (VDPV), the use of oral polio attenuated live Vaccine OPV must be stopped in the future to completely eradicate polio. Only by using the inactivated polio vaccine IPV can VAPP and VDPV risks be avoided. The inactivated poliomyelitis vaccine can prevent poliomyelitis IPV outbreak, can generate colony immunity and is the best choice for eliminating subsequent stages of poliomyelitis. At present, manufacturers are in the world to produce IPV, but the production virus strains are wild strains. The production of IPV by using wild strains needs to meet the requirement of biological safety level 3 (BSL-3), and the condition of even BSL-4 is not absolutely guaranteed after the poliomyelitis is eliminated all over the world. For the Vaccine manufacturers to have difficulty in meeting such strict biological safety requirements, currently, the currently approved DTaP-IPV/Hib combined Vaccine on the market in China is only PentaximTM produced by SanofiPasteur (SP) company, in the combined Vaccine, the strain selected by the IPV Vaccine is Salk strain, but the Salk strain belongs to a strong wild strain and has strong toxicity, so that the WHO encourages the Vaccine manufacturers to develop attenuated strain poliomyelitis inactivated Vaccine Sabin IPV (inactivated poliovirus Vaccine, Sabin strain, abbreviated as sIPV). To date, most of the strains selected for IPV in multivalent combination vaccines that are marketed globally are the Salk strains. Therefore, the development of a combined vaccine containing the sIPV with higher safety and stability is of great significance.

Haemophilus influenzae (Haemophilus influenzae, Hi) is by far the leading causative agent of invasive diseases in humans, most of which are also caused by Haemophilus influenzae type b (Hib). Hib meningitis is the first of bacterial meningitis, and has the characteristics of high morbidity, high fatality rate and high disability rate; it is also characterized by small onset age, high infant infection rate, and the onset age mainly focuses below 5 years, especially below 2 years. Worldwide, an estimated 300 million serious diseases are caused each year, about 38.6 thousands of people die, and the disease becomes a global public health problem. Hib pneumonia plays an important role in childhood infectious diseases in developing countries, accounting for about 20% of all childhood pneumonia, and is an important cause of death in developing countries. In the early 90 s of the last century, Hib conjugate vaccines are incorporated into conventional children's immunity programs in countries such as Western Europe and the United states, and the incidence of Hib invasive epidemic diseases is rapidly reduced or disappeared. Currently, the World Health Organization (WHO) is working on driving its inclusion into the planned immunization programs of other countries. In China, the level of Hib natural infection antibodies in children of 3-5 years old is low, and the Hib natural infection antibodies are high risk groups. Clinical research in hospitals in China suggests that Hib meningitis accounts for 51.7% of the children with suppurative meningitis, and 84% of the children are under 2 years old; hib pneumonia accounts for 34.3% in children, so that the necessity of Hib conjugate vaccination is urgently needed to be solved in China.

Due to the distribution difference of epidemic diseases and the limitation of research difficulty, the Hib component in similar products developed at home and abroad at present is still prepared into corresponding combined vaccine by adopting a freeze-drying process; the freeze-dried vaccines sold in the market need to be redissolved before immunization, and the redissolution process is complicated; meanwhile, based on the safety risk and the limitation of capacity introduced by the freeze-drying process, the research of the liquid combined vaccine of the absorbed acellular pertussis-poliovirus inactivated vaccine-b-type haemophilus influenzae (combined) is carried out, and the method has good social value and economic value.

Disclosure of Invention

The purpose of the present invention is to provide a liquid vaccine composition that can prevent pertussis, diphtheria, tetanus, poliomyelitis, and Haemophilus influenzae type b at the same time.

The 5 infectious diseases are still the important causes of infant death until now, and are brought into the state EPI, and 12 injections are needed after inoculation (Baikukou: 3 times of basic immunity and 1 times of boosting immunity; poliomyelitis: 3 times of basic immunity and 1 times of boosting immunity; Haemophilus influenzae type b: 3 times of basic immunity and 1 times of boosting immunity). In order to improve the condition that the inoculation times are various and avoid wrong inoculation and missed inoculation, the liquid vaccine composition provided by the invention can simultaneously prevent the liquid vaccine composition of the infectious diseases, and the liquid vaccine composition is inoculated for 4 times (3 times of basic immunization and 1 time of boosting immunization) according to the inoculation procedure. Greatly reducing the workload of inoculation and the burden of children.

Specifically, the invention provides a liquid vaccine composition, which is a liquid and comprises: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus Sabin strain I, inactivated poliovirus Sabin strain II, inactivated poliovirus Sabin strain III, a haemophilus influenzae type b capsular polysaccharide-protein conjugate.

The invention also provides a liquid vaccine composition, which is liquid, wherein the antigen consists of the following substances: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus Sabin strain I, inactivated poliovirus Sabin strain II, inactivated poliovirus Sabin strain III and a haemophilus influenzae type b capsular polysaccharide-protein conjugate.

According to the invention, the Sabin strain has the advantage of high safety, in order to ensure the immunogenicity of the Sabin strain and avoid the interference effect of the Sabin strain and other antigens in the combined vaccine as much as possible, the antigen proportion of the poliovirus of the Sabin strains of type I, type II and type III in the composition is preferably (4-15): (11-45): (11-45). As a particularly preferred embodiment of the present invention, the following components are included in each 0.5mL of the liquid vaccine composition: sabin type I strain 4-15 DU of poliovirus, Sabin type II strain 11-45 DU of poliovirus, and Sabin type III strain 11-45 DU of poliovirus. Experiments show that when the antigen proportion of the type I, type II and type III Sabin strain poliovirus in the liquid vaccine composition is 1: (2.5-3.5): (2.5-3.5), the liquid vaccine composition has an optimal immune effect as a whole.

The haemophilus influenzae type b capsular polysaccharide-protein conjugate is formed by conjugating haemophilus influenzae type b capsular polysaccharide with physiologically acceptable carrier protein. The physiologically acceptable carrier protein is selected from one or more of diphtheria toxoid, nontoxic variant of diphtheria toxin CRM197, tetanus toxoid, meningococcal outer membrane protein.

The vaccine composition provided by the invention can further comprise one or more of an aluminum adjuvant, a soluble phosphate buffer solution, sodium chloride and a stabilizer on the basis of the antigen stock solution. In particular, adjuvants, such as adjuvants comprising aluminium hydroxide and/or aluminium phosphate; practice shows that when the aluminum content is 0.3-1.25 mg/ml, preferably 0.4-0.8 mg/ml, the titer of each type of antigen can be kept at a higher quality standard while the safety of the liquid vaccine composition is ensured. The vaccine composition may also include a quantity of soluble phosphate buffer to maintain the pH within a reasonable range (e.g., pH 5.8-7.2), and a quantity of sodium chloride or equivalent substances to maintain osmotic pressure. The stabilizer can be selected from vitamins and/or amino acids, preferably amino acids; the invention discovers that the stability of the polysaccharide-protein conjugate of the haemophilus influenzae type b in a liquid preparation can be obviously improved by adding amino acid into the liquid composition, so that the overall stability of the liquid composition is improved. The stabilizer can also be selected from an M199 culture medium; practice shows that when 5-20% of 10X M199 culture medium concentrated solution is added into the combined vaccine, the stability of the poliovirus antigen is remarkably improved, so that the stability of the whole liquid vaccine composition is significantly improved.

As a preferred embodiment of the present invention, the liquid vaccine composition comprises the following components:

the liquid vaccine composition provided by the invention can be used for basic immunity and boosting immunity.

The invention discovers through a great deal of practice that when the proportion and the dosage of the pertussis toxin, filamentous hemagglutinin and pertactin contained in the composition are optimally adjusted, the vaccine composition can further meet the requirements of basic immunity or enhanced immunity on the basis of ensuring the safety.

When the liquid vaccine composition is used for preparing a basic immune vaccine, the mass ratio of pertussis toxin, filamentous hemagglutinin and pertactin contained in the composition is preferably (20-25): (20-25): (3-8). Preferably, the composition contains 20-25 mug of pertussis toxin, 20-25 mug of filamentous hemagglutinin and 3-8 mug of pertactin per 0.5ml single dose. Further preferably, the composition contains 25 μ g of pertussis toxin, 25 μ g of filamentous hemagglutinin and 8 μ g of pertactin per 0.5ml single dose.

As a preferred embodiment of the present invention, the liquid vaccine composition can be used for basic immunization, and the following components are contained in the composition per 0.5ml single dose:

when the liquid vaccine composition is used for preparing a booster vaccine, the mass ratio of pertussis toxin, filamentous hemagglutinin and bordetella pertussis adhesin contained in the composition is preferably (2.5-8): (5-8): (2.5-3). Preferably, the composition contains 2.5-8 mug of pertussis toxin, 5-8 mug of filamentous hemagglutinin and 2.5-3 mug of pertactin per 0.5ml single dose. Further preferably, the composition contains 8 μ g of pertussis toxin, 8 μ g of filamentous hemagglutinin and 2.5 μ g of pertactin per 0.5ml single dose.

As a preferred embodiment of the present invention, the liquid vaccine composition can be used for boosting immunity, and the following components are contained in each 0.5ml single dose of the composition:

the invention also provides a method for preparing the liquid vaccine composition.

The method specifically comprises the following steps: mixing liquid raw materials including pertussis toxin, filamentous hemagglutinin, pertactin, diphtheria toxoid raw liquid, tetanus toxoid raw liquid, inactivated Sabin I strain poliovirus raw liquid, inactivated Sabin II strain poliovirus raw liquid, inactivated Sabin III strain poliovirus raw liquid and b type haemophilus influenzae polysaccharide-protein conjugate raw liquid. The compositions provided by the present invention are liquid formulations, wherein the various raw materials are also prepared in liquid form and then directly mixed.

After the liquid raw materials are mixed, substances such as an adjuvant, a soluble phosphate buffer solution, sodium chloride, a stabilizer and the like are further added, so that a stable liquid vaccine composition suitable for human bodies can be obtained.

The invention protects the application of the liquid vaccine composition in the preparation of basic immunity vaccines.

The invention protects the application of the liquid vaccine composition in preparing a booster vaccine.

The invention provides a vaccine kit comprising a first chamber and a second chamber; the first chamber contains a basic immunity vaccine prepared from the liquid vaccine composition; the second chamber contains a booster vaccine prepared from the liquid vaccine composition.

The invention provides a vaccine kit, comprising a first chamber, a second chamber and a third chamber; the first chamber and the second chamber respectively contain basic immunity vaccines prepared by the liquid vaccine composition; the third chamber contains a booster vaccine prepared from the liquid vaccine composition.

The invention provides a vaccine kit, which comprises a first chamber, a second chamber, a third chamber and a fourth chamber; the first chamber, the second chamber and the third chamber respectively contain basic immunity vaccines prepared by the liquid vaccine composition; the fourth compartment contains a booster vaccine prepared from the liquid vaccine composition.

During actual inoculation, the vaccine kit provided by the invention can efficiently complete the vaccination, and avoid wrong inoculation or missed inoculation. For example: the basic immunization is carried out by using the basic immunization vaccine in the first chamber (or the first chamber and the second chamber are used in sequence, or the first chamber, the second chamber and the third chamber are used in sequence) in the kit, and then the boosting immunization is carried out by using the boosting immunization vaccine in the second chamber (or the third chamber or the fourth chamber) in the kit.

Compared with the prior art, the vaccine composition provided by the invention is a liquid preparation, can maintain the stability of the property of the liquid preparation in each link of preparation, transportation, storage and use, avoids the safety risk and the limitation of capacity caused by the introduction of a freeze-drying process, simultaneously avoids the complicated re-dissolving process of the freeze-dried vaccine before immunization, and reduces the production and use costs. Furthermore, the vaccine composition provided by the invention can be used for basic immunization or boosting immunization, and can improve the titer of the antibody in the serum of an immunized person on the basis of ensuring the safety.

Drawings

FIG. 1 shows the results of type I, type II, and type III neutralizing antibody titer assays for sIPV according to the immunization procedure described in example 8;

FIG. 2 shows the results of the pertussis (aP) antibody titer assay according to the immunization procedure described in example 8; wherein, aP-PT represents pertussis toxin, aP-FHA represents filamentous hemagglutinin, aP-PRN represents pertactin;

FIG. 3 shows the results of Diphtheria (DT), tetanus (TT) antibody titer assays according to the immunization procedure described in example 8;

FIG. 4 shows the results of the Hib antibody titer assay according to the immunization program described in example 8.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

This example provides a liquid vaccine formulation comprising the following components in an amount per 1 human dose:

8 mug of pertussis toxin, 8 mug of filamentous hemagglutinin, 2.5 mug of pertactin, 2Lf of diphtheria toxoid, 5Lf of tetanus toxoid, 7.5DU of inactivated poliovirus type I stock solution, 22.5DU of inactivated poliovirus type II stock solution, 22.5DU of inactivated poliovirus type III stock solution, 10 mug of haemophilus influenzae type b polysaccharide-protein conjugate, 0.6mg/ml of aluminum, a proper amount of sodium chloride aqueous solution, and the pH value of phosphate buffer is adjusted to 5.8-7.2. The vaccine may also be supplemented with appropriate amount of stabilizer, such as M199 culture medium and/or amino acids.

The liquid vaccine formulations provided in this example can be used to boost immunity.

Example 2

8 mug of pertussis toxin, 8 mug of filamentous hemagglutinin, 2.5 mug of pertactin, 2Lf of diphtheria toxoid, 5Lf of tetanus toxoid, 15DU of inactivated poliovirus I stock solution, 45DU of inactivated poliovirus II stock solution, 45DU of inactivated poliovirus III stock solution, 15 mug of haemophilus influenzae b-protein conjugate, 0.8mg/ml of aluminum, a proper amount of sodium chloride aqueous solution, and the pH value of phosphate buffer is adjusted to 5.8-7.2. The vaccine may also be supplemented with appropriate amount of stabilizer, such as M199 culture medium and/or amino acids.

Example 3

25 mug of pertussis toxin, 25 mug of filamentous hemagglutinin, 8 mug of pertactin, 12.5Lf of diphtheria toxoid, 3.5Lf of tetanus toxoid, 15DU of inactivated poliovirus type I stock solution, 45DU of inactivated poliovirus type II stock solution, 45DU of inactivated poliovirus type III stock solution, 10 mug of haemophilus influenzae type b polysaccharide-protein conjugate, 0.8mg/ml of aluminum, and the pH value of a sodium chloride aqueous solution is adjusted to 5.8-7.2 by a proper amount of phosphate buffer. The vaccine may also be supplemented with appropriate amount of stabilizer, such as M199 culture medium and/or amino acids.

The liquid vaccine formulations provided in this example can be used for basic immunization.

Example 4

25 mug of pertussis toxin, 25 mug of filamentous hemagglutinin, 8 mug of pertactin, 12.5Lf of diphtheria toxoid, 3.5Lf of tetanus toxoid, 7.5DU of inactivated I-type poliovirus stock solution, 22.5DU of inactivated II-type poliovirus stock solution, 22.5DU of inactivated III-type poliovirus stock solution, 15 mug of b-type haemophilus influenzae polysaccharide-protein conjugate, 0.8mg/ml of aluminum, a proper amount of sodium chloride aqueous solution, and a pH value adjusted to 5.8-7.2 by phosphate buffer. The vaccine may also be supplemented with appropriate amount of stabilizer, such as M199 culture medium and/or amino acids.

Example 5

This example provides a method for preparing each of the monovalent vaccine stock solutions described in examples 1 to 4.

1. Diphtheria toxoid stock solution

Opening diphtheria bacillus, transferring 1-3 generations of diphtheria bacillus in a seed tube of a toxin production culture medium, transferring the diphtheria bacillus to the toxin production culture medium to prepare seeds for production, culturing the seeds by using a culture tank liquid, culturing the seeds for 45-52 hours at 34-36 ℃ by using a maltose comprehensive culture medium, adding formaldehyde to sterilize after the culture is finished, adding ammonium sulfate to precipitate impure protein into a culture, centrifugally collecting supernatant, performing ultrafiltration concentration, adding ammonium sulfate to salt out and precipitate diphtheria toxin protein, centrifugally collecting precipitate, adding PB buffer solution to dissolve, performing ultrafiltration to remove ammonium sulfate in toxin, adding formaldehyde, standing for 35-37 ℃, detoxifying for 30 days, performing ultrafiltration to remove formaldehyde in toxoid, and performing sterilization filtration to obtain a diphtheria toxoid raw liquid.

2. Tetanus toxoid stock solution

Starting clostridium tetani, transferring 1-3 generations of clostridium tetani in a seed tube of a toxin production culture medium, transferring to the toxin production culture medium to prepare seeds for production, culturing the seeds by adopting a culture tank liquid, culturing the seeds by adopting a double-aged culture medium at 34-36 ℃ for 62-72 hours, adding formaldehyde for sterilization, filtering the culture solution to remove a sterilization body, adding ammonium sulfate to precipitate toxin protein, centrifugally collecting precipitate, adding formaldehyde after removing ammonium sulfate in toxin by ultrafiltration, standing for 35-37 ℃, detoxifying for 30 days, removing formaldehyde in toxoid by ultrafiltration, and performing sterilization filtration to obtain tetanus toxoid stock solution.

3. Acellular pertussis vaccine stock solution

1) The method comprises the steps of starting bordetella pertussis, inoculating the bordetella pertussis to an improved bag-ginger culture medium or an activated carbon semi-integrated culture medium, culturing at 35-37 ℃ for no more than 48 hours, inoculating the bordetella pertussis to a CPB culture medium for two generations, culturing for 18-26 hours per generation, performing amplification culture to prepare enough production seeds, inoculating the seeds to a fermentation tank for liquid culture, culturing for two days at 37 ℃ by using the CPB culture medium, adding thimerosal or formaldehyde into a culture after the culture is finished for sterilization, centrifuging the harvested culture, and storing the thalli and supernatant obtained by centrifugation at 2-8 ℃ for further purification.

2) Purification of pertussis toxin and filamentous hemagglutinin: ultrafiltering and concentrating the supernatant, then loading the supernatant into a Capto SP chromatographic column, eluting a mobile phase by using a urea-containing phosphate buffer solution in a sodium chloride gradient manner, respectively collecting elution components containing pertussis toxin and filamentous hemagglutinin, loading the elution components containing pertussis toxin into a Capto MMC chromatographic column, eluting the buffer solution by using a Tris-HCL buffer solution in a sodium chloride gradient manner, and collecting a pertussis toxin elution peak; and (4) loading the elution peak containing the filamentous hemagglutinin to a hydroxyapatite chromatography column for purification, and collecting the elution peak.

3) Separation and purification of pertussis adhesin: releasing the thallus at 60 ℃, performing ultrafiltration concentration, loading the thallus to a Captoadhere chromatographic column, collecting an elution component containing the pertussis adhesin by taking an acetic acid buffer solution as a mobile phase, loading the thallus to a CaptoSp chromatographic column, performing gradient elution by taking the acetic acid buffer solution as the mobile phase and sodium chloride, and collecting an elution peak, namely the separated and purified pertussis adhesin.

4) Preparing acellular pertussis stock solution: detoxication of pertussis toxin by glutaraldehyde, treatment of filamentous hemagglutinin and pertussis adhesin by formaldehyde, degerming and filtering to obtain acellular pertussis stock solution.

4. Poliovirus stock solution

Poliovirus type I stock solution:

1) cell preparation: taking 1 or more cell tubes in a working cell bank, recovering the cell tubes, then culturing the cell tubes in a first-stage cell reactor at 36.5 +/-0.5 ℃ until the cell concentration is 1-5 multiplied by 106 cells/ml; digesting the cells in the first-stage cell reactor by trypsin, inoculating the cells to the second-stage cell reactor, and culturing at 36.5 +/-0.5 ℃ until the cell concentration is 2-10 multiplied by 106 cells/ml; digesting the cells in the second-stage cell reactor by trypsin, inoculating the cells to a third-stage cell reactor, culturing at 36.5 +/-0.5 ℃ until the cell concentration is 1-5 multiplied by 106 cells/ml, and inoculating the viruses.

2) Virus culture and harvest: and replacing the cell culture solution in the third-stage cell reactor with a maintenance solution, inoculating the I-type poliovirus working seed batch virus seeds into Vero cells according to the MOI of 0.002-0.02, and culturing at 32.5 +/-0.5 ℃. Culturing the virus for 2-4 days, and harvesting cell supernatant, namely the monovalent harvesting solution of the poliovirus Sabin strain.

3) Clarifying and purifying a virus harvest solution: the harvest liquid is concentrated by 50-200 times by using a 300-and 500-kDa ultrafiltration membrane after centrifugal clarification. And after the concentration is finished, performing centrifugal purification and separation on the virus by adopting a sucrose density gradient, wherein the sucrose gradient is specifically a sucrose density gradient solution with a first gradient of 30% sucrose and a second gradient of 55% sucrose, centrifuging at 30000rpm for 10 hours, and collecting a zone where the virus is located. And (3) carrying out anion exchange chromatography on the virus liquid collected by density gradient centrifugation, monitoring the wavelengths of 280nm and 254nm, and collecting flow-through liquid to obtain virus chromatography liquid. And concentrating the virus chromatography liquid to obtain a purified liquid. The anion exchange chromatography may be DEAE Sepharose Fast Flow, Q Sepharose Fast Flow or other anion exchange chromatography. In one embodiment of the invention, the anion exchange chromatography used is DEAE Sepharose Fast Flow.

4) Virus inactivation: sterilizing and filtering the purified solution with the particle size of 0.22 mu m, adding a formaldehyde solution to ensure that the final concentration of free formaldehyde is 90 mu g/ml, inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃, and continuously inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃ after sterilizing and filtering to obtain the monovalent stock solution.

Poliovirus type II stock solution:

2) Virus culture and harvest: and replacing the cell culture solution in the third-stage cell reactor with a maintenance solution, inoculating the II-type poliovirus working seed batch virus seeds into Vero cells according to the MOI of 0.01-0.1, and culturing at 32.5 +/-0.5 ℃. Culturing the virus for 2-4 days, and harvesting cell supernatant, namely the monovalent harvesting solution of the poliovirus Sabin strain.

Poliovirus type III stock solution:

2) Virus culture and harvest: and replacing the cell culture solution in the third-stage cell reactor with a maintenance solution, inoculating Vero cells with the batch virus seed MOI of the III-type poliovirus working seeds of 0.05-0.5, and culturing at 32.5 +/-0.5 ℃. Culturing the virus for 2-4 days, and harvesting cell supernatant, namely the monovalent harvesting solution of the poliovirus Sabin strain.

5) Virus inactivation: sterilizing and filtering the purified solution with the particle size of 0.22 mu m, adding a formaldehyde solution to ensure that the final concentration of free formaldehyde is 90 mu g/ml, inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃, and continuously inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃ after sterilizing and filtering to obtain the monovalent stock solution.

5. Haemophilus influenzae type b polysaccharide-protein conjugate vaccine stock solution

1) Starting a working seed batch strain of the haemophilus influenzae type b, inoculating the working seed batch strain to a Hib comprehensive culture medium, culturing for 16-24 hours at the temperature of 35-37 ℃ in a 5-8% carbon dioxide environment, then performing third-generation amplification culture to prepare seeds for production, inoculating the seeds to a fermentation tank for culture at the temperature of 35-37 ℃ for 6-12 hours, and adding formaldehyde for sterilization; centrifuging sterilized culture solution to collect supernatant, adding cetyl trimethyl ammonium bromide to the final concentration of 1.0g/L, fully stirring, standing overnight, centrifuging to collect polysaccharide, stirring the precipitated polysaccharide with sodium chloride solution for 3 hours to dissociate the polysaccharide and the cetyl trimethyl ammonium bromide, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 25% (v/v), standing overnight at 2-8 ℃, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 75-80% (v/v), fully shaking to precipitate the polysaccharide, standing overnight, centrifuging to collect precipitate, washing with absolute ethanol and acetone for three times respectively, and drying to obtain crude polysaccharide; dissolving crude sugar in 10% saturated central sodium acetate solution, adding cold phenol solution according to the proportion of 1:1(v/v), oscillating, mixing uniformly, centrifuging, collecting supernatant, extracting for 1-3 times, clarifying the supernatant, collecting the supernatant, removing residual phenol and nucleic acid by ultrafiltration membrane ultrafiltration, adding 4mol/L sodium chloride solution into the polysaccharide solution after ultrafiltration until the final concentration of sodium chloride is 0.3mol/L, adding 95% ethanol until the final concentration of ethanol is 75% (v/v), mixing uniformly, standing overnight at 2-8 ℃, precipitating refined sugar, centrifuging, collecting precipitate, sequentially adding absolute ethanol and acetone, washing twice, and drying to obtain the refined sugar.

2) Dissolving refined sugar in water for injection, and mixing the obtained solution with polysaccharide: cyanogen bromide solution is added according to the proportion of 1:0.5(w/w), the temperature is maintained at 23 +/-3 ℃, the pH value is 10.8 +/-0.2, reaction is carried out for 30 minutes, and after activation is finished, the ratio of polysaccharide: adding oxalyl hydrazine solution of 2.8 percent in the proportion of 1:3.5(w/w), keeping the temperature at 23 +/-3 ℃, reacting for 15 minutes at the pH of 8.5 +/-0.2, performing derivatization, clarifying and filtering, and performing ultrafiltration by using 0.05mol/L sodium chloride to remove cyanogen bromide, namely obtaining polysaccharide derivatives; according to the polysaccharide: calculating the dosage of tetanus toxoid according to the ratio of protein to protein of 1: 0.8-1.2 (w/w), adding the tetanus toxoid into a polysaccharide derivative solution, and mixing the tetanus toxoid: adding carbodiimide into carbodiimide at a ratio of 1:10(w/w), maintaining the reaction temperature at 5 +/-3 ℃ and pH at 5.7 +/-0.2, reacting for 60 minutes, adjusting the pH to 6.9 +/-0.1 to stop the reaction, purifying the polysaccharide protein conjugate by using a Sepharose 4FF gel chromatography column, monitoring the absorption value of OD280nm by using 0.15mol/L sodium chloride solution as a mobile phase, collecting an elution peak near V0, combining eluates obtained by each chromatography to obtain a purified conjugate, and sterilizing and filtering to obtain the Haemophilus influenzae type b conjugate vaccine stock solution.

Example 6

The embodiment provides a preparation method of the liquid vaccine composition in the embodiments 1 to 4, which comprises the following steps:

1) mixing pertussis toxin, filamentous hemagglutinin, pertussis adhesin, diphtheria toxoid stock solution, tetanus toxoid stock solution, inactivated poliovirus type I stock solution, inactivated poliovirus type II stock solution, inactivated poliovirus type III stock solution and haemophilus influenzae type b combined vaccine stock solution provided in example 5, adding an aluminum salt adjuvant diluted by a sodium chloride aqueous solution, adding a phosphate buffer solution and a stabilizer, and adjusting the pH to 5.8-7.2 to obtain a semi-finished product;

2) and (3) subpackaging the semi-finished product with a penicillin bottle or a prefilled syringe.

Example 7

The embodiment provides a finished product inspection method of the liquid vaccine preparation provided in the embodiment 1-4.

1 identification test

The detection of PT, FHA and PRN antigens by enzyme-linked immunosorbent assay should contain corresponding antigens (appendix of Baibaishao combined vaccine adsorption in the three parts of '2015 edition' of Chinese pharmacopoeia). By adopting the immune double diffusion method (the three parts of the 2015 edition of the Chinese pharmacopoeia), the product should form an obvious precipitation line with diphtheria toxoid, tetanus toxoid, haemophilus influenzae type b immune serum and tetanus toxoid immune serum.

2 physical examination

2.1 appearance: after shaking, the mixture should be in the form of uniform milky white suspension without non-scattering clots or foreign bodies

2.2 loading: checking according to law, and the content should not be less than the indicated amount (not less than 0.5 ml).

3 chemical examination

3.1pH value: should be 5.8 to 7.2.

3.2 aluminum content: should not be higher than 0.8 mg/dose.

3.3 free Formaldehyde content: should not be higher than 0.2 g/L.

3.4 glutaraldehyde content: should be less than 0.01 g/L.

3.5 polysaccharide content: d ribose is used as a reference, the content of polysaccharide in the test sample is calculated according to the content of ribose, and the dosage of the polysaccharide for 1 time of human use is 10-15 mu g.

4 potency assay

4.1 acellular pertussis vaccine: the dilution is carried out to the first immunization dose by using a proper dilution factor, and then the dilution is carried out according to a 5-fold series. The immunization time was 21 days. The dose for 1 time of human use should have an immune titer not less than 4.0IU, and the 95% confidence limit should be not less than 2.0 IU. If the above requirements are not met, a retest can be performed, but all valid test results must be calculated as geometric means (e.g., using weighted geometric means when using probability analysis). If the requirements are met, the product is judged to be qualified.

4.2 diphtheria vaccine: the immune titer of diphtheria toxoid should be not less than 30IU per 1 human dose.

4.3 tetanus vaccine: the immunization titer of the tetanus toxoid in each 1 human dose should be not less than 40 IU.

5 efficacy test

5.1 Haemophilus influenzae type b conjugate vaccine

Each batch of vaccine was injected subcutaneously with 10 NIH (or BALB/c) mice weighing 12-14 g, and another 10 mice from the same batch were used as controls and injected with 0.85% sodium chloride solution. Injecting Hib conjugate vaccine containing 2.5 μ g polysaccharide twice subcutaneously on days 1 and 14, collecting blood via retroorbital vein on days 21-28, measuring anti-Hib IgG antibody by ELISA method, and determining Cutoff value by absorbance value of mouse serum of 0.85% sodium chloride solution control group, wherein the serum anti-Hib IgG antibody level of the vaccine group should be higher than Cutoff value of more than 80% mice.

5.2 adsorption of Sabin Strain inactivated poliomyelitis vaccine

After serial gradient multiple dilution is carried out on each batch of vaccine, Wistar rats with the concentration of 180-220 g above the cleaning level are immunized respectively, 10 dilution degrees are provided for each group, the male and female parts are half, and each animal is immunized with 0.5ml of test sample. Collecting blood for 21 days, separating serum, and storing at-20 deg.C. And (3) respectively detecting the titer of the neutralizing antibody of the anti-I/II/III type 3 viruses in the serum, and calculating the seroconversion rate. The efficacy test of the internal reference substance is carried out by the same method. The test article should be comparable in immunogenicity to the internal reference, i.e.: test article ED₅₀The value is not higher than 400% of the reference.

6, sterility inspection: the bacteria should be grown aseptically according to the examination by law.

7 pyrogen examination

According to legal inspection, 1.0 mu g of polysaccharide is injected into each lkg of rabbit body weight, and the body temperature rise of three preliminarily tested rabbits is lower than 0.6 ℃, and the body temperature rise of 3 rabbits is lower than 1.3 ℃ comprehensively; or in 5 re-tested rabbits, the temperature of the rabbits with the temperature rise of 0.6 ℃ or higher than 0.6 ℃ does not exceed 1 rabbit, and the total temperature rise of 8 rabbits in the initial test and the re-test is 3.5 ℃ or lower than 3.5 ℃, which all judge that the pyrogen examination of the test sample meets the regulation.

8 specific toxicity test

8.1 acellular pertussis vaccine: the toxic reference was diluted as indicated for each batch. NIH mice (female or male half) with the weight of 14-16 g are used, and each dilution of the toxicity reference substance and the test substance are used as one group, and each group contains at least 10 mice. Each mouse was injected with 0.5ml of the intraperitoneal solution, and the a-b tests were performed.

a) Mouse leukocytosis assay

Peripheral blood of the mice was collected 3 days after the injection to count leukocytes. The test result is statistically processed, and the leukocytosis activity of the mouse injected with the test article is not higher than 0.5 LPU/ml.

b) Mouse histamine sensitization test

4 days after injection, 0.5ml of a solution (containing 4mg of histamine dihydrochloride or 2mg of histamine diphosphate) was intraperitoneally injected into each mouse, and 30 minutes later, the anal temperature of each mouse was measured. The test result is statistically processed, the activity of the histamine sensitization toxicity of the mice of the test article is not higher than 0.8HSU/ml, and no animal is dead.

8.2 diphtheria, tetanus vaccines

The guinea pigs weighing 250-350 g were used, each batch was injected subcutaneously into the abdomen for 2.5ml, each batch was injected bilaterally for 1.25ml, and the observation was carried out for 30 days. The injection site can be infiltrated and become hard after 5-10 days, and may not be completely absorbed after 30 days. Weighing body weight on day 10, day 20 and day 30, increasing body weight before injection, and treating patients without topical suppuration, necrosis, tetanus and late stage paralysis.

9 toxicity reversal experiments: each test batch was placed at 37 ℃ for 4 weeks, following protocol 8.1 b).

10 bacterial endotoxin test: according to the general rule 1143 of three parts of the Chinese pharmacopoeia, the dose should be no higher than 100 EU.

The results of the tests are shown in Table 1.

Table 1: vaccine test results

Example 8

This example provides a method of performing mixed immunization using the liquid vaccine formulation provided by the present invention. The method specifically comprises the following steps:

the liquid vaccine formulation provided in example 4 was used as a base immunization formulation vaccine and injected subcutaneously twice on days 1 and 14, respectively. Blood was collected from the orbit on day 28, and the IgG antibody titer in the mouse serum was measured by indirect ELISA. After 3 months (i.e., day 90), blood was collected again to assay the IgG titer in the serum, and the liquid vaccine preparation provided in example 1 was injected subcutaneously as a booster, and the IgG titer in the serum was assayed at 14 days (i.e., day 104) after the booster. The results are shown in Table 2 and FIGS. 1 to 4.

Table 2: results of antibody titer detection

In FIGS. 1 to 4, P is < 0.05; p < 0.01; represents P < 0.001.

The experimental results show that the vaccine liquid preparation provided in example 4, after being immunized twice as a basic immunizing agent, can induce mice to produce significant neutralizing antibodies against poliovirus type I/II/III, antibodies (IgG) against pertussis (aP) components (PT, FHA, PRN), antibodies (IgG) against DT, antibodies (IgG) against TT, and antibodies (IgG) against Hib. After 3 months, we found that there was a significant reduction in the level of antibody in mice, neutralizing antibodies against poliovirus type I/II/III, antibodies against pertussis components (PT, PRN) (IgG), and antibodies against DT (IgG). However, after the vaccine liquid preparation provided in example 1 is used as a booster for immunization once, the neutralizing antibody against poliovirus type I/II/III, the antibodies (IgG) against pertussis components (PT, FHA, PRN), the antibodies (IgG) against DT, the antibodies (IgG) against TT and the antibodies (IgG) against Hib in mice can be significantly improved.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A liquid vaccine composition, wherein the composition is a liquid comprising: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus Sabin strain I, inactivated poliovirus Sabin strain II, inactivated poliovirus Sabin strain III, a haemophilus influenzae type b capsular polysaccharide-protein conjugate.

2. A liquid vaccine composition, wherein said composition is a liquid and wherein the antigen comprises: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus Sabin strain I, inactivated poliovirus Sabin strain II, inactivated poliovirus Sabin strain III and a haemophilus influenzae type b capsular polysaccharide-protein conjugate.

3. The composition according to claim 1 or 2, wherein the composition has a ratio of the type I, type II and type III Sabin strains poliovirus antigens of (4-15): (11-45): (11-45), preferably 1: (2.5-3.5): (2.5-3.5).

4. The composition according to claim 1 or 2, wherein the haemophilus influenzae type b capsular polysaccharide-protein conjugate is formed by conjugating haemophilus influenzae type b capsular polysaccharide with a physiologically acceptable carrier protein;

the physiologically acceptable carrier protein is selected from one or more of diphtheria toxoid, nontoxic variant of diphtheria toxin CRM197, tetanus toxoid, meningococcal outer membrane protein.

5. The composition according to any one of claims 1 to 4, wherein the composition further comprises one or more of an aluminum adjuvant, a soluble phosphate buffer, sodium chloride and a stabilizer;

preferably, the stabilizer is one or more of vitamins, amino acids and M199 culture medium.

6. The composition according to any one of claims 1 to 5, wherein the mass ratio of pertussis toxin, filamentous hemagglutinin and pertactin contained in the composition is (20 to 25): (20-25): (3-8);

preferably, the composition contains 20-25 mug of pertussis toxin, 20-25 mug of filamentous hemagglutinin and 3-8 mug of pertactin per 0.5ml single dose.

7. The composition according to claim 6, wherein a single dose of said composition per 0.5ml comprises the following components:

8. the composition according to any one of claims 1 to 5, wherein the pertussis toxin, filamentous hemagglutinin, and pertactin are contained in the composition in a ratio of (2.5 to 8): (5-8): (2.5-3);

preferably, the composition contains 2.5-8 mug of pertussis toxin, 5-8 mug of filamentous hemagglutinin and 2.5-3 mug of pertactin per 0.5ml single dose.

9. The composition according to claim 8, wherein a single dose of said composition per 0.5ml comprises the following components:

10. a process for preparing a liquid vaccine composition according to any one of claims 1 to 9, comprising the steps of: mixing liquid raw materials including pertussis toxin, filamentous hemagglutinin, pertactin, diphtheria toxoid raw liquid, tetanus toxoid raw liquid, inactivated Sabin I strain poliovirus raw liquid, inactivated Sabin II strain poliovirus raw liquid, inactivated Sabin III strain poliovirus raw liquid and b type haemophilus influenzae polysaccharide-protein conjugate raw liquid.

11. Use of a liquid vaccine composition according to any one of claims 1 to 7 for the preparation of a basic vaccine.

12. Use of a liquid vaccine composition according to any one of claims 1 to 5, 8 or 9 for the preparation of a booster vaccine.

13. A vaccine kit comprising a first chamber and a second chamber; the first chamber contains a basic immunity vaccine prepared by the liquid vaccine composition of any one of claims 1 to 7; the second chamber contains a booster vaccine prepared from the liquid vaccine composition of any one of claims 1 to 5, 8 and 9;

or, a first chamber, a second chamber, and a third chamber; the first chamber and the second chamber respectively contain a basic immunity vaccine prepared by the liquid vaccine composition of any one of claims 1 to 7; a third chamber containing a booster vaccine prepared from the liquid vaccine composition of any one of claims 1 to 5, 8 and 9;

or, a first chamber, a second chamber, a third chamber, and a fourth chamber; the first chamber, the second chamber and the third chamber respectively contain a basic immune vaccine prepared by the liquid vaccine composition of any one of claims 1 to 7; the fourth chamber contains a booster vaccine prepared from the liquid vaccine composition according to any one of claims 1 to 5, 8 and 9.